Medium for production of penicillin



Patented pt 2, 1,952 1 Arneen's tamemf'rene mete; iiiigassigniii' t6 'Tii'tl.,'a"c6rp6iatfoniif Mar'yrana I, No-Drawing, ApplicationApril-io, 1948 V Serial 'No. 20,373

in e ta ee la e t Tpenicilli'r'i- P ands-more particularly toaife rinehtat' eularly useful f orwenie'il'li n. premier t; iswell known that penicillin is firbdu n1 recoverable quantities by the. growth of. certain microorganisms; such. as Penicill iuih. 'hdtat'lim and Penieillium chrys-ogenum The rennenta; tion medium wherein penicillin-producing orgam'sms are 'grown ordinarily comprises enema nutrient which has dissolved or suspended therein a fermentable carbohydrate and a suitable sb'iiice of' nitrogen. If desired; other" materials may be added to the nutrient medium,' "such as ee'i ta in'mineralsalts which may be reqlnrectforth g r'owth of the organism, or pH regulatory materiars, suchas calcium carbonate; In addi* ti'o'r i it is wen recognized that the amount of activenenicinimn the'completely'fermentedbeer" ma be increased by theadd-itionfof certainad juva'nts, such; as pnenyia'cetamiae; enema '01 f ii-iiring'iheferfirehtatierr process.-

T Ini the corni'n'e'i'eial manufacture of penicillin by fermentation methods, the nutr ent medium which most gen-emu empioredatthe-present actosea's tnerermentan-e v I I Marmara-m1- e'dntaihed" in 'st'eeb water as source or nitrogen for: the penrciu'impmde irig' near or eettbnseednour ma eemployee in.

plate ersteep wat r to supply the nitrogen nee; s'sarifi'fiTtfi full grown; and: eevempmerit or substituting eet'tonseedmeei" for steep water and when calb'ium carbonate mueatormfiamtn, results: achieved are at least' seam toendqs'amenmes superior" to the results ea 11 mentation even though the amount of penicillin KM 1 M enema recoyer the 139111011 prese nutrient The dilii my,

jected to further extraction processes for the recoverybf penicillin therefrom. In view of this recovery difficulty, it was not considered that cottonseed meal could be used in a commercial process for the preparation of penicillin by fer in the fermented beer may be somewhat increased when compared with the amounts 01 penicillin produced when using steep water as the r1m'ary source of nitrogen in the nutrient me ium.-

Accordingly it is one object of this invention to provide a nutrient medium for nenicillin grobepre 'areu-ri-emrea'clily'availablemateha MA still further obj lc of this provisionof a process fort d cillin by rementatienw cillin" are increase myeereedflyreeevere diam; ,1 h E1111 jed i from the" following adee'ribtidn and tended elem;

. 1 3 trose produced by Corn Products Refining Company of Argo, Illinois), or a partially dextrinized starch. If desired, calcium carbonate may be added to the medium in order to control the pH 4 to be poor if the solids were resuspended in the mother liquor prior to filtration.

As indicated in Table I a series of five fermenters were set up. To each fermenter was of the fermenting medium and suitable adju- 5 added 200 grams of lactose, 100 grams of calcium vants may be employed if desired. One satiscarbonate and 3 grams of phenylacetamide as an factory adjuvant which has been extensively emadjuvant. To each of fermenters No. 1 and No. 3 ployed inftheart is phenylacetamide. was added the filtrate obtained from the water As previously indicated, serious recovery difextraction of 300 grams of Profio prepared as ficultie with respect to initial filtration are enoutlined above. To each of fermenters No. 2 and countered when cottonseed meal or cottonseed No. 4 were added the water-extracted residues of flour is employed as such as the nitrogen source Profioleft on the N0. 615 paper as above indiin a, fermentation medium for penicillin procated. To fermenter No. 5 was added 300 grams duction. For this reason, even though the of untreated Profio. Each fermenter was then amounts of penicillin produced during the fermade up to ten liters with water and it will be mentation process may be increased, such a menoted that the nutrient medium in each ferdium could not be used commercially. In accordmenter was the same except with respect to the ance with this invention, however, it has been Profio fraction employed. After sterilization of discovered that the filtrability of the fermented the medium the fermenters were each inoculated medium may be markedly-improved if the cottonwith a strain of Penicillium designated as New seed meal or flour or the soybean meal or fiour Wisconsin Q-l'TG which had previously been is first subjected to a water extraction step and propagated for 24 hours in a bran medium. The ifthe resulting water-extracted residue is used five fermenters were incubated under the same to prepare the fermentation medium. When this conditions at about 24 C. and air was passed isidone the filtrability of the fermented mate- 25 through the fermenters at the rate of one volrial to separate the beer is markedly improved. ume/volume/minute. Agitation of each ferment- In'addition it ha been discovered that the averer was effected with turbine blades revolving at age yield of penicillin is increased when" the 400 R. P. M. The fermenters were assayed pewater-extracted residue is employed in place of riodically for penicillin. The results of this exthe meal or flour which has not been subjected periment are clearly indicated in Table I.

Table I Medium Assays, u./m1.

Fermentor 3 days 4 days 6 days No Profio Prollo Fraction pm sun pm am pm 61 87 93 104 119 214 455 440 580 551 3 49 81 9a 120 109 4- Insoluble of300 g 256 403 519 689 720 620 a 300 100 126 244 an 436 494 to the water extraction procedure. While it might be expected that the water extract contains the substance that would promote growth and the formation of penicillin in the fermented medium, such was not foundto be the case. and greatly, superior results are obtained when the residue is employed in the medium as compared to when the water extract itself is employed in the medium. In order to demonstrate the findingsin this respect and to demonstrate the un-' expected advantages obtained in practicing this invention as outlined above, the following experiment was carried out, the resultsbeing shown in-Table I. V

I A commercial cottonseed fiour intended for human consumption and sold under the trade name Proflo was selected. This material is one mm'wmcn. the cottonseed oil had previously been extracted by heat and pressure and it is sold by Traders Oil Mill Company of Fort Worth, Texas. In order to remove the water soluble fraction of this cottonseed hour for these particular experiments, the following method was employed 300 grams of Profio were weighed into all; liter Erlenmeyer flask. Four-liters of'tap water were added, mixed thoroughly and the mixture was autoclaved at 1209C. for two hours. The solids were allowed. to settle out and the mother liquor was then filtered through No. 615 paperv on a Buchner'funnel, precaution being taken to decant the liquid first. Filtration was satisfactory bythis method but had a tendency It will be noted that fermenters No. 2 and No. 4 show considerably higher assays of penicillin expressed as units per milliliter than the assays of fermenters No. 1 and No. 3, thus clearly demonstrating that the water-extracted residue of cottonseed meal is considerably more active in stimulating the formation of penicillin in the fermentation medium than the water soluble portion. This is the inverse of what might be expected. It will be further noted in connection with the assay analyses given in Table I that at the five day fermentation period, both in the morning (a. m.) and afternoon (p. m.) the penicillin yield was significantly higher in fermenters No. 2 and No. 4 than in No. 5. This is important because in'commercial practice fermenters are run'for a period of four days. In addition, after fermentation was completed, the fermentation liquid from fermenter No. 5 was difiicult to filter in order to separate the beer from the mycelia of the micro-organism. This difiiculty was not experienced in fermenters No. 2 and No.. 4, thus demonstrating further improved results that are obtained when employing the water-extracted residue of the cottonseed'fiour.

The foregoing data clearly shows the improved results that are obtained when employing the water-extracted residue of cottonseed flour in accordance with one embodiment of this invention.

In order to demonstrate the improved results obtainable by using water-extracted residues of cottonseed fiour and'soybean meal as compared wait-t e whole;flouraonnneal. imafidor'dancenwlth;

finyention, ataseriesszof .zfermenters:

up;.aeach -of-twhichlwas:preparemjinoculated: and incubated :in the; mannenoutlined'm the; previous experiment;With-theeexceptions notedzmf-Tablefl.

to which reference is heremade. .7 i

necti'onJmei-th the preceding; experiments; concentrations of the several ingredients Lin. the; medium-are;notsrcriticaL;howeyerpbuteshoulmfbe maintainedmithinzthe ranges willzmccogniz'edzin: theirzartzafor. :the zipreparationz of a fermentation;

mediumiiorapenicillinproduction;

Foamedover. l y .I I As indicated in this 'table', there -werenseven fer menters set up. Each fermenter"contairred lo liters ofmedium yand had dissolved Qrsuspend'ed therein 2 percentlactose, '-1 per cent calcium -car-' bonat'e and-l03yper cent phenylacetamide. Fermenters No. land-No; 2"contained 3 percent of solvent-extractedsubstantially oiL-free soybeanmeal from a commercial source and fermenter No. 3 contained a water-extracted residue ofthe soybean meal. "The-water-extracted residue was prepared as previously indicated in connection with Proflo. :Fermentersz-No;=4zzand No. 5 contained 3. pe r cent whole Proflo flour and fermentersNo. '6 'and'No; '7 contained the waterclearly indicates that improved IQSMESIEIZBJOQQR tamed, 'whe remplqy ng .the water: ins0lub1e=;fra0

tien instead qfathe. whole.;-flour Inzzadditionsthek. fermented medium containing ithe water-sin.

soluble fraction was much more readily filtered in order to separate the mycelia from the beer.

With reference to fermenters No. 1 through No. 3 containing the soybean material, it will be noted that the results clearly show that the medium prepared from water-extracted soybean meal has a higher assay than that prepared from whole soybean meal which had not been water-extracted.

The foregoing experiments summarized in Tables I and II clearly demonstrate that it is possible to improve penicillin yields by employing a fermentation medium containing the water-extracted residue of cottonseed flour or soybean meal. In commercial practice these substances may be employed in whole or in part to replace the steep Water which has been used in commercial fermentation processes. A suitable fermentation medium for use on a commercial scale preferably contains the water-extracted residue, lactose, calcium carbonate and phenylacetamide in the proportions indicated in con- 'Itw'ill', of course,'be understood thateithefcob" q s d m a or cq onsee n maY-b e e preparing the fermentatiommedium"and lilrewise either soybean meal or soybean flour inalfxi similarly beused; 'The'term iriealas-employ V Y in the accompanying claims "is intended to -als'o' includeflour: An important considerationis'fthaj'fi the'-meal or flour 'bepreviouslysubjected to a water-extraction treatment before incorporation?- mt the fermentation medium; v f;

It "is :preferred to employ a cottonseed*mealior. a cottonseed flourif rom*which thenaturafoil hasi beenremoved by solvent extraction arty-meat and pressure treatment. 'Ifiike'wise' if soybean meal or flour is employed; it is' "preferred to .remove theoilfsothat'the'meal issubstantiallyoili free; This is" desired, parti-cularlyjbecauseof" economic considerations and" also because the" oilmay have an adverse effect upcn-the-ultimate recoveryof-crystalline penicillin. from thefermentation' beer'. V 1

In preparingthe fermentation-'mediumi 'this ief carried out by any of'the -methods that" are well? known in the art andf-prior tofermentationthe; medium is "preferably sterilized in accordan'cej with the practice well known to those skilled it the art. Likewise the inoculation of "the""'com= mercial' fermenter is a wellknown" procedure" which does not form a part of this-invention: While lactose- --is the preferred 'fermentable carbohydrate foruse in connection withthemedium of this invention, other fermentable carbohydrates, of course, may be employed either alone or in combination with the lactose. Suitable fermentable carbohydrates that may be used in connection with penicillin-producing strains of organisms are glucose and partially dextrinized starch. The calcium carbonate employed in the medium is for the purpose of controlling the acidity of the fermenting medium.

The phenylacetamide of the medium functions primarily as an adjuvant to increase the amount of penicillin formed without actually materially afiecting the growth of the organism. The function of these adjuvants is not clearly understood but it is believed that they are directly involved in the chemical reactions which take place during the synthesis of the penicillin by the organism. While phenylacetamide is preferred, other adjuvants may be employed, such as 2-phenylethylamine, p-hydroxy phenylethylamine, phenylacetic acid, phenylethylamine hydrochloride, phydroxyacetamide, phenoxyacetic acid, p-chloro-- phnylaceticlacid, p-bromo' and p-i'odophem' yla'cetic acid, etch.

After thexfermentatio'n has been effected by the organism in; the medium of this invention, the

penicillin may be recovered by any of the methods that are well knownin'the art. It has been:

found that the medium of this invention does not I adversely affect the commercial recovery of 7 crystalline penicillin.

While several particular embodiments of this invention are shown above, it will be understood, of course, thattheinvention isnot to be limited thereto, since many modifications may be made, and it is contemplated, therefore, by the appended claims, to cover any such modifications 8. producing" microorganism; a nutrient medium" comprising .water,' an .assimilable carbohydrate, and the water-insoluble residue remaining after water extraction"atwabout 120 .C. of a, member selected from the group consisting of cottonseed meal and soybean meaL- J 7. In a method for the production of penicillin 'by fermentation of a nutrient medium with a penicillin-producing microorganism, the improvement which consists of carrying out said fermentation in a nutrient medium containing as an essential ingredient the water-insoluble residue from water-extraction of a meal selected from the group consisting of cottonseed meal and soy-,

as, fall within thetrue spirit and scope of this inyention.

What is claimed is: 1

j 1. ;A fermentation medium for penicillin productioncomprisifig water, an assimilable carbohydrate, and the water-insoluble residue remaining after water extraction of a member selected fromthe-group consisting of cottonseed meal soybean meal. 2.. A .fermentatidn'medium for penicillin produotion comprising vvater, an assimilable carbohydratIan adjuv'ant selected from the group consisting of phenylacetamide, 2-phenylethylamine,

p;hydroxyphenylethylamine, phenylacetic acid,

phenylethylamine hydrochloride, p-hydroxyacetamidejphenoxyacetic acid, p-chlorophenylacetic 5. Afermentation' medium for penicillin pro-.

duction comprising Water, an assimilable carbohydrate', and the water-insoluble residue remainirig after water-extraction at about 120 C. of a member selected from the group consisting of cottonseed meal and soybean meal.

I 6. A method forthe production of penicillin which comprises fermenting with a penicillinbean meal.

8. A method. for the production of penicillin which comprises-fermenting-with a penicillinproducing microbrganism..a nutrient frnediiimf comprising water, an assimilabl'e" carbohydratei and a water-insoluble residuefrom water-extrac tion of a meal selected from the group consisting of cottonseed meal and soybean meal.

9. The-method recited in claim 8 wherein-said meal iscottonseedmeal. 4 v we 10. A method for the production- 0f penicilli which comprises fermenting ,with a penicillin: producing microorganism a nutrient medium comprising water, lactose,- calcium carbonate; phenylacetamide, and a substantially oilfree water-insoluble residue of water-extracted cottonseed meal. i

V ALFRED R. STANLEY.

REFERENCES orrEo I g The following references areof'record in"'the1 file of this patent:

UNITED STATES ATENTS Number 2,051,017 Schwarz Aug. 11,1936 2,437,918 McCormack Mar. 16, 1948 2,448,790 Foster et a1. jsept. 7,1948 2,918,791 Foster et al. Sept. 7, 1948 2,470, 07 ,Moyer e July 12, 1949 v OTHER REFERENCES Watts et all, Indaand Eng. Chem, Oct. 1939, page1282.

'Foster et a1, Jr. 'Bacteri0logy, Vol.51,--No. 4, April 1946;page's 474-476. V I

Woodr'uff et al;, A Study of Protein Eatract from Soybeans, Jr.' -Agri. Research, vol. 57, Nov.

15,1938, pages 737, 738 and 743. 

7. IN A METHOD FOR THE PRODUCTION OF PENICILLIN BY FERMENTATION OF A NUTRIENT MEDIUM WITH A PENCILLIN-PRODUCING MICROORGANISM, THE IMPROVEMENT WHICH CONSISTS OF CARRYING OUT SAID FERMENTATION IN A NUTRIENT MEDIUM CONTAINING AS AN ESSENTIAL INGREDIENT THE WATER-INSOLUBLE RESIDUE FROM WATER-EXTRACTION OF A MEAL SELECTED FROM THE GROUP CONSISTING OF COTTONSEED MEAL AND SOYBEAN MEAL. 